Activation of the innate immune system induces the production of cytokines, costimulatory molecules and antigen-presenting molecules that enhance the induction of antigen-specific adaptive immunity. Activation of the innate immune system also induces the production of antimicrobial products that may directly combat infectious agents. The innate immune system is activated by molecules that interact with specific toll-like receptors (TLR). Many cells express TLR, including macrophages and dendritic cells that reside throughout the host. Cells at the interface of the host and the environment such as mucosal epithelial cells, mucosal dendritic cells, and gamma-delta TCR T cells also express TLR. Activation of the innate immune system at the time of vaccination with select agent antigens may enhance the induction of antigen-specific protective immunity. Activation of the innate immune system before exposure to select agent pathogens may increase host resistance due to the production of antimicrobial products. Dimerized single chain antibodies reactive with specific TLR developed in other projects will be tested for their ability to activate the innate immune system as determined by their ability to enhance the induction of antigen-specific immunity (antibody, Th-1, Th-2 cells, Tc-1, Tc-2) and their ability to reduce morbidity and mortality after bacterial, viral or toxin challenge. We will also determine if the administration of TLR-specific single chain antibodies to immunized mice at the time of challenge enhances protection against bacterial, viral or toxin pathogenesis by activation of the innate immune system. The specific aims of this project are: Specific Aim 1: Develop anti-TLR polyclonal sera in mice (for use in other projects to develop anti-TLR scFv). Specific Aim 2: Develop systemic and aerosol challenge models for Yersinia pestis, vaccinia and anthrax lethal toxin. Specific Aim 3: Evaluate anti-TLR scFv tetramers as systemic and mucosal adjuvants when conjugated to the model antigens HIV-1 gp120 and ovalbumin. Specific Aim 4: Evaluate anti-TLR scFv-diabodies as systemic and mucosal adjuvants when used as fusion proteins with Yersinia V-antigen, vaccinia B5R and anthrax protective antigen. Specific Aim 5: Evaluate anti-TLR scFv diabody-antigen fusion proteins for their ability to protect mice against lethal Yersinia, vaccinia or anthrax lethal toxin challenge.